Biocompatibility and colorectal anti-cancer activity study of nanosized BaTiO3 coated spinel ferrites

In the present work, different nanoparticles spinel ferrite series (MFe2O4, Co0.5M0.5Fe2O4; M = Co, Mn, Ni, Mg, Cu, or Zn) have been obtained via sonochemical approach. Then, sol–gel method was employed to design core–shell magnetoelectric nanocomposites by coating these nanoparticles with BaTiO3 (BTO). The structure and morphology of the prepared samples were examined by X-ray powder diffraction (XRD), scanning electron microscope (SEM) coupled with energy dispersive X-ray spectroscopy (EDX), high-resolution transmission electron microscope (HR-TEM), and zeta potential. XRD analysis showed the presence of spinel ferrite and BTO phases without any trace of a secondary phase. Both phases crystallized in the cubic structure. SEM micrographs illustrated an agglomeration of spherical grains with nonuniformly diphase orientation and different degrees of agglomeration. Moreover, HR-TEM revealed interplanar d-spacing planes that are in good agreement with those of the spinel ferrite phase and BTO phase. These techniques along with EDX analyses confirmed the successful formation of the desired nanocomposites. Zeta potential was also investigated. The biological influence of (MFe2O4, CoMFe) MNPs and core–shell (MFe2O4@BTO, CoMFe@BTO) magnetoelectric nanocomposites were examined by MTT and DAPI assays. Post 48 h of treatments, the anticancer activity of MNPs and MENCs was investigated on human colorectal carcinoma cells (HCT-116) against the cytocompatibility of normal non-cancerous cells (HEK-293). It was established that MNPs possess anti-colon cancer capability while MENCs exhibited a recovery effect due to the presence of a protective biocompatible BTO layer. RBCs hemolytic effect of NPs has ranged from non- to low-hemolytic effect. This effect that could be attributed to the surface charge from zeta potential, also the CoMnFe possesses the stable and lowest zeta potential in comparison with CoFe2O4 and MnFe2O4 also to the protective effect of shell. These findings open up wide prospects for biomedical applications of MNPs as anticancer and MENCs as promising drug nanocarriers.

www.nature.com/scientificreports/ as a property of a fast cellular growing state [4][5][6] . The depolarized membrane potential makes tumor cells more susceptible to electroporation, permitting the delivery inside the cells through the produced pores 7 . The generated electric field by MENCs can be variated through many parameters one of them is the type of magnetic phase (core) in core-shell MENCs. Barium titanate, BaTiO 3 (noted BTO), is a smart material that exhibits a piezoelectric characteristic through the generation of electrical polarization in response to minute structural deformations 8 . It has been stated that BTO possesses biological characteristics including high biocompatibility when contacted with biological cells. Therefore, it has been considered as a promising material in biomedicine applications 9 . Ciofani et al. have reported the cytocompatibility of BTO NPs at higher concentrations such as 100 μg/ml on mesenchymal stem cells (MSCs) 10 . According to Ref. 11 , poly(lactic-co-glycolic) acid/BTO NPs have shown their role in cell attachment and the effects on the differentiation and proliferation of osteoblast and osteocytes.
Spinel ferrite is the most attractive group of iron oxide materials due to the diversity in the chemical composition leading to a broad range of physical characteristics in a variety of applications [12][13][14][15] . The structure of spinel ferrite consists of a cubic close-packed arrangement of oxygen ions with total 56 atoms that are subdivided into 32 O 2− anions and 24 cations. The spinel ferrite structure possesses two crystallographic sites where 8 A-sites are occupied by tetrahedrally coordinated cations and 16 B-sites are octahedrally coordinated 16 . The spinel magnetic properties are governed by the type of metal cations and their distribution between the two crystallographic sites 17,18 . The metal cations distribution is affected by several factors including the ionic radii of cations, size of the interstitial site, stabilization energy, preparation method, and the reaction conditions 19 . The magnetic materials are divided based on their capability to be magnetized and demagnetized. In general, there are two types of magnetic materials which are hard and soft magnets. Hard magnets retain permeant magnetization in the absence of an applied field, while soft magnets are easy to magnetize and demagnetize.
Magnetic nanoparticles possess a considerable interest in biomedical applications for diagnosis and cancer therapy 20 . Magnetic nanoparticles are capable to act as a drug delivery system 21,22 where it accumulates at the tumor sites through passive or active targeting. Passive targeting mostly relays on exploiting the enhanced permeability and retention (EPR) effect, due to the leaky nature and physiologically defective tumor vasculature as well as the lack of a lymphatic system for drainage 23 . On contrary, active targeting is based on the magnetic response of nanoparticles via applied magnetic fields. Hyperthermia is another cancer therapy technique where the cancer cells can be destroyed when subjected to high temperatures (40-45 °C) [24][25][26][27] . Magnetic nanoparticles produce heat when exposed to an alternating magnetic field due to relaxations of rotating magnetic moment 20 . Moreover, magnetic nanoparticles have been utilized as enhanced contrast agents in magnetic resonance imaging (MRI) 28 .
The potential practical bio-applications of nanoparticles can be considered only when their toxicity is very well understood. In particular, each time a new nanomaterial aimed for biomedical applications required an extensive examination of its biosafety. Hemolysis is a considerable blood compatibility analysis as the nanoparticles could be directly contacted with red blood cells (RBC) via bloodstream injection. Hemolysis occurs when the RBC membrane is damaged, leading to leakage of hemoglobin. This causes several adverse health effects such as renal toxicity, hypertension, and anemia. Furthermore, the other blood compartments [platelets and white blood cells (WBC)] can be also affected through intravascular hemolysis which leads to coagulation, or immune deficiency 29,30 36 . The nanoparticlescell interaction can be initiated by adhering the nanoparticles to the cell surface, then are internalized via endocytosis, and amassed inside digestive vacuoles. Thus, it is very likely to happen cytotoxicity at higher concentrations due to particle overload to the cells 32 .
To our knowledge, there is no evidence has been found in the literature on bioactivities examination of core-shell (MFe 2 O 4 @BTO, Co 0.5 M 0.5 Fe 2 O 4 @BTO; where M = Mn, Ni, Mg, Cu, Zn, or Co) MENCs on human colorectal cancer (HCT-116) and human embryonic kidney (HEK-293) cell lines. Thus, this study aims to confirm that MNPs and MENCs do not impact harmful effects on healthy cultured cells and do not promote the growth of cancer cells. We have prepared MNPs and MENCs by sonochemical and sol-gel synthesis approaches, respectively. The surface and structural characterizations were investigated through XRD, SEM, EDX, TEM, and zeta potential procedures. Next, the preliminary in vitro assessment of cytocompatibility and cell viability have been conducted through MTT assay, nuclear DAPI staining, and hemolysis analysis on HCT-116, HEK-293, and RBCs with a special focus on the protective properties of BTO on the used cells.

Results and discussion
XRD structural analysis. Figure 1 represented the XRD patterns of prepared spinel ferrite (CoFe 2 O 4 , CoMnFe) MNPs and core-shell MENCs (MFe 2 O 4 @BTO, CoMFe@BTO; M = Mn, Ni, Mg, Cu, Zn, or Co). The XRD exhibited the pure spinel ferrite and core-shell structure without any trace of impurity phases. It demonstrated the characteristic peaks of spinel planes for (CoFe 2 O 4 , CoMnFe) which are indexed as (220), (311), (222), (400), (422), (511), and (440). The recorded peaks of the spinel were well-matched with the cubic structure and space group Fd-3m of spinel ferrite according to card No. 96-591-0064 [37][38][39][40] Fig. 2A. Samples exhibit nonuniformly diphase orientation (bright and medium dark regions) of agglomerated spherical grains. It is difficult to completely disperse the core material despite the vigorous ultrasonication dispersion of MNPs in BTO precursor solution during the coating process. Thus, they   Fig. 2B. The EDX spectra emphasized the existence of the elements without any trace of impurities indicating the purity of the prepared samples. The TEM images stressed the formation of the core dark region (spinel ferrite MNPs phase) and the surrounding bright shell (BTO phase) as shown in Fig. 3. It can clearly distinguish the interface between two phases in the TEM images. The variation of core-shell color is due to the difference in transmission intensity and electron penetration efficiency on MNPs and BTO 43 . Moreover, the MNPs form agglomerates in BTO matrix. The corresponding highresolution transmission electron microscopic (HR-TEM) images illustrate the well-defined lattice fringes of the magnetic core and BTO shell. The moire patterns are dominant in HR-TEM images which clearly exhibit the interference of crystallographic orientations of the ferrite and BTO phases. The crystallography of the two phases was proved by calculating the interplanar d-spacings that are in good agreement with planes of the ferrite phase and planes of the BTO phase. The interface between spinel ferrite and BTO phases is clearly shown by HR-TEM. Therefore, at this interface, the movement of strain between the ferrite and ferroelectric phase could happen and it might be suitable to build a strong ME coupling in the core-shell nanocomposite.
Zeta potential measurements. The zeta potential is a valuable technique for assessing surface charge on the nanoparticles, predicting their stability and inferring the state of the surface 44 . Usually, nanoparticles having zeta potential in the range − 10 to + 10 has a neutral charge, while a zeta value higher than + 30 mV or lower than − 30 mV indicated a highly anionic and cationic surface respectively 38 . The zeta potential of MNPs and MENCs was studied and summarized in Table 2. It is clear from the zeta potential results that MnFe 2 O 4 has the highest zeta potential as compared to other MNPs and MENCs, followed by CoFe 2 O 4 . CoMnFe showed the lowest zeta potential. Furthermore, results indicated that MNPs and MENCs have cationic surfaces 45 .   www.nature.com/scientificreports/ ions under the influence of various hydrolyzing enzymes in the phagolysosomes at low pH as well as the proteins participating in iron metabolism and utilizing according to natural iron metabolism pathways 47,48 . Nevertheless, the degradation of CoFe 2 O 4 within lysosome leads to slow etching and releasing of cobalt ions Co 2+ where it is known to be toxic in larger doses 49,50 . Moreover, the cytotoxicity could be attributed to the ionization of metallic NPs inside the cells known as "Trojan-horse" mechanism according to Hsiao et al. 51 . Earlier studies have also     Generally, we have observed that MENCs either maintain the cell viability or promote the cell proliferation within the certain composites. This may be related to the presence of BTO shell. It is a piezoelectric nanomaterial and possesses an ability to act as an active substrate to promote cellular growth under physiological environment 9 . BTO can generates an electric stimulation as response to transient structure deformation due to the migration and attachment of cells 8 . The generated electrical pulses are transmitted to the surrounding cells which promotes the cell signaling pathways and stimulates Ca 2+ -calmodulin pathway that responsible for synthesis the growth factor and enhance the cell growth 56,57 . G. Genchi et al. used BTO NPs to promote tissue regeneration. They have shown that the presence of BTO NPs in the scaffold was able to enhance the growth rate and proliferation of H9c2 myoblasts after 72 h 58 . BTO is the most promising nanomaterial with huge potential in a wide range of nanomedicine applications. Owing to its good biocompatibility, protectivity and its applicability in multifunctional theranostic systems including drug delivery, cell stimulation, and tissue engineering 58 . www.nature.com/scientificreports/ The Impact of MNPs and MENCs on nuclear morphology. The quantitative study was further augmented via the qualitative analysis of the cell nuclear morphology visualization under confocal microscope using DAPI (4′,6-diamidino-2-phenylindole) staining. It is fluorescent stain that binds very strongly to DNA and appears to associate with A-T rich regions in minor grove 59 . The passing of DAPI through live cell is less efficiently and therefore the effectiveness of the stain is low, thus cell must be permeabilize or fixed for the DAPI to enter the cell and bind with DNA. DAPI is normally used for cell counting, measuring apoptosis, and nuclear segmentation tool in high conducting imaging analysis. In this report, the colorectal carcinoma HCT-116 cells were stained with DAPI to visualize the impact of MNPs and MENCs on nuclear DNA. Also, it was used to identify the number of nuclei, visualization the apoptosis characteristic features include chromatin condensation, nuclear shrinkage and fragmentation, and to assess the gross cell morphology 31,60 . Figure  Erythrocyte lysis assay. The hemolytic potential assay has been conducted to assess the toxicity of different MNPs and MENCs formulations at the cellular level as illustrated in Figs. 10 and 11. According to ISO 10993-4 which stands for the blood compatibility evaluation of the medical devices contain or generate nanomaterials. The standard states the following criteria of hemolysis percentage where (0-2%) is nonhemolytic biomaterial, (2-5%) slightly hemolytic, or (> 5%) hemolytic 29 . It has been observed that all the formulation in this study at the lowest concentration 33 µg/0.  www.nature.com/scientificreports/ lytic effect (0-2%). In contrast, the highest concentration 276 µg/0.1 ml exhibited a slightly to high hemolytic effect (> 5%) as detailed in Table 3 and Fig. 12. Upon close analysis, the presence of a biocompatible BTO layer plays a crucial role in terms of reducing the hemolytic effect of different core formulations even with the highest concentration as shown in Fig. 12. The large surface-to-volume ratio is one of the most important parameters of NPs where the smaller size of particle, the larger surface area they have. Although NPs possess the advantage of large loading drug due to large surface area, however; they promote the reaction of oxygen with tissues and creating free radicals 47 which is oxidative stress factor on the cell. It has been acknowledged from literatures that the cytotoxicity and human cells apoptosis are generally based on the ROS production and oxidative stress due to the exposing to MNPs [61][62][63] . Several studies reported that the blocking of nanoparticles ROS leads to minimize their interaction with RBCs membrane and therefore their potential hemolytic effect 64 . Therefore, uncoated MNPs might be cytotoxic due to the direct contact with cells 65 .
Comparison in the biological activities of MNPs and MENCs. The NPs cytotoxicity and adverse hematology effect depend on various particle parameters. The main influencing factors are materials' morphology, size, composition, hydrophobicity, surface area, and surface charge 29 . On the other hand, different biological parameters influence cytotoxicity like cell type, culture and exposure conditions (i.e. cell density, particle concentration, and temperature 66 . In addition to oxidative stress, the other mechanisms of toxicity and forms of injuries might be resulted from NPs interaction include protein denaturation, membrane damage, DNA damage, and immune reactivity 67 . Or analysis of lysosomes membrane which lead to leaking of analytical enzymes into the cell resulting in cell apoptosis 68 . The obtained hemolysis and cytotoxicity results are summarized in Table 3. Commonly, inverse structure magnetic ferrite exhibited an obvious reduction in cell viability, while normal structure magnetic ferrite showed an opposite action through maintaining the cell viability or promoting the cellular growth. These findings can be explained by the spinel ferrite MNPs activity where it depends on different parameters such as particle size, surface texturing, stability, metal ions redox properties, and cations distribution among tetrahedral and octahedral sites 69 . CoFe 2 O 4 belongs to inverse spinel ferrites were Fe 3+ have tetrahedral coordination and (Co 2+ ) and (Fe 3+ ) are equally distributed in octahedral sites 70 . The spinal's ferrite MNPs surfaces www.nature.com/scientificreports/ mainly composed of octahedral sites. According to the previous reports, the metal ions that occupied the octahedral positions play a crucial role in the catalytic activity due to the longer bond length; thus, it can be easily interact with the reactant molecules 69,71,72 . However, the metal ions that occupied the tetrahedral sites are rarely contributed to the reduction activity. The inactivity of this crystallite coordinate site can be originated from the strong metal-oxygen bonds because of the lower valency and coordination number. Furthermore, the tetrahedral cations are not freely accessible to the reactants 73 Table 3. Moreover, CoMnFe MNPs has shown the nonhemolytic effect even at highest concentration 276 µg/0.1 ml, while the CoFe 2 O 4 MNPs at the same concentration exhibited slightly hemolytic effect. This can be attributed to the different catalytic action of simple and mixed magnetic ferrite which is correlated to the electronic structure as well as the synergic interaction between different metals 74 . Moreover, this could be correlated to the surface charge from zeta potential measurements in Table 2 where the CoMnFe possesses the stable and lowest zeta potential in comparison with CoFe 2 O 4 and MnFe 2 O 4 . Once we got a homogenous metal solution, the pH was arranged equally to 11 by using 2 M NaOH solution. The sonication probe (Ultrasonic homogenizer UZ SONOPULS HD 2070 with a power of 70 W and a frequency of 20 kHz) was used to conduct the reaction for 1 h. The obtained product was washed several times with hot deionized water. Then it was dried at 180 °C for 12 h and crushed in an agate mortar to get MNPs.

Materials and methods
Preparation of core-shell MENCs. The citrate sol-gel auto-combustion procedure was used to prepare MENCs. Firstly, 1.9 g of barium carbonate was mixed with 10 ml of deionized water and 10 ml of ethanol with continuous stirring for 20 min. Similarly, 2.8 ml of titanium (IV) isopropoxide was mixed with 50 ml ethanol and 50 ml deionized water with continuous heating and stirring at 80 °C temperature and 30 min, respectively. In a separate beaker, these two prepared solutions were mixed then 4.2 g citric acid was added and placed on a hot plate at (80 °C) with stirring for 20 min. The as-prepared MNPs were dispersed in 20 ml of ethanol using a sonication bath for 30 min at room temperature. Later, the MNPs suspension was mixed with prepared BTO precursor solution and then placed in the sonication bath for vigorous vibration at 80 °C for 2 h. Finally, the resultant product was retained on the hot plate at 80 °C and kept until the solution becomes thick white near to gel. Then, the temperature was raised to 120 °C to burn the formed gel. Subsequently, the received powder was grounded and then calcined in a muffle furnace at 800 °C for 5 h to obtain core-shell MENCs powder. Figure 13 illustrates the schematic sequence of the experimental procedure.  Erythrocyte lysis assay. The erythrocyte lysis assay was conducted according to Shivashankarappa et al. 76 .
The spectrophotometer was utilized to examine the cytotoxicity by the measuring the amount of hemoglobin released via RBC's membrane rupture. The fresh blood was taken from adult wistar rat and EDTA was added to the collecting tube to prevent blood coagulation. It was centrifuged for 10 min at 1500 rpm at 4 °C and the plasma www.nature.com/scientificreports/ with white layer containing WBC and platelets was removed carefully by aspiration. Thereafter, the erythrocytes pellets were washed three times with PBS (pH 7.4) and resuspended in PBS to give nine times its volume. Two different concentrations (lowest 33 µg/0.1 ml and highest 267 µg/0.1 ml) of MNPs and MENCs were used for RBCs treatment and the PBS was added to reach the total volume of 2 ml. Then, it was incubated for 20 min at 37 °C followed by centrifuging at 2000 rpm for 3 min. The supernatant was collected, and the density of the color measured at 540 UV visible spectrophotometer. 1% SDS was used as a positive control, and the PBS was used as a negative control. The percent of hemolysis was calculated according to the following formula 77 : Statistical analysis. All statistical analyses were run on GraphPad Prism Software [Version 9.0].
Mean ± standard error (S.E.M) from control, MNPs and MENCs was calculated. One-way analysis of variance (ANOVA) with Dunnett's post hoc test were used to calculate the difference between control and NPs treated groups. Error bars ± S.E.M. *p < 0.05; **p < 0.01; ***p < 0.001 versus control.
Consent for publication. All authors have read and agreed the final draft of the manuscript for consideration for publication.

Conclusions
In the present study, we have used sonochemical and sol-gel techniques to prepare various (MFe 1.8 O 4 , CoMFe) MNPs and core-shell (MFe 1.8 O 4 @BTO, CoMFe@BTO,) MENCs. XRD analysis confirmed the purity of all products (MNPs and MENCs) and the average crystallite size of core-shell MENCs which was evaluated within 24-45 nm range. The morphology analyses (both TEM and SEM) revealed the aggregated spherical grains with different agglomeration degree with various spinel ferrite magnetic core. Core-shell MENCs were designed to overcome the disadvantages that associated with MNPs in term of physical and biological enhancement. It was proved that magnetic core coated with BTO matrix is biocompatible. Moreover, the usage of MENCs in cancer therapy do not require heat generation which could potentially damage the surrounding healthy tissue. They can efficiently release drug in controlled protocol independent of physiological changes in the presence of magnetic field. We have also evaluated the biological impact of (MFe NCs and MnFe 2 O 4 revealed a toxic effect for both cell lines while the CoMnFe NCs exhibited the selective anticancer action on colorectal cancer cells due to the metals' synergic effect and the electronic structure differences. Consequently, the CoNiFe NCs possess a highly toxic effect for both cell lines thus it is not recommended in biomedical applications. The coating of MNPs with biocompatible BTO layer reduce the pro-apoptotic effect of magnetic core. MENCs eliminated the direct contact of uncoated MNPs with cells, therefore it relived the toxicity of MNPs. RBCs hemolytic effect of NPs has ranged from non-to low-hemolytic effect. This effect that could be attributed to the surface charge from zeta potential the CoMnFe possesses the stable and lowest zeta potential in comparison with CoFe 2 O 4 and MnFe 2 O 4 . Also, to the protective effect of shell. Further examinations are required to investigate the cellular effect in different incubation time, concentrations, and to ensure the cytocompatibility and carcinogenicity of MNPs and MENCs. This study was conducted and applied on in vitro, so applying it in future in vivo studies is highly recommended. Developing a high quality magnetoelectric materials, with suitable structure, morphology, particles size, surface charges and minimum denaturation with the lowest cytotoxic effect is a demanding plan for anti-cancer drugs and drug carriers. So, using certain formulations with BTO is a promising strategy targeting cancer.

Data availability
All data generated or analyzed during this study are available within this manuscript.